长链非编码RNA HCG4通过Wnt/β-catenin通路对垂体腺瘤细胞增殖、迁移与侵袭的影响

Effects of long chain non-coding RNA HCG4 on proliferation, invasion and migration of pituitary adenoma cells through Wnt/β-catenin signaling pathway

目的探讨长链非编码RNA HLA复合体4(HCG4)调控Wnt/β-连环蛋白(β-catenin)通路对垂体腺瘤细胞增殖、迁移和侵袭生物学行为的影响。方法实时荧光定量PCR(qPCR)检测35对垂体腺瘤和正常垂体组织的HCG4水平。培养垂体腺瘤HP75细胞并分为Control组(无转染)、NC组(转染空载体pcDNA3.1)、HCG4组(转染过表达质粒pcDNA3.1-HCG4)和HCG4+Wnt激动剂组(同时转染过表达质粒pcDNA3.1-HCG4和Wnt激动剂SKL2001)。CCK-8法、划痕实验和Transwell小室实验分别检测HP75细胞的增殖、迁移和侵袭的情况。qPCR和Western blotting检测HP75细胞的β-catenin和基质金属蛋白酶(MMP)-9水平。结果垂体腺瘤组织的HCG4水平低于正常组织(0.616±0.044 vs.1.000±0.071,P<0.05);21例Ⅰ~Ⅱ期垂体腺瘤患者(0.712±0.053)和14例Ⅲ~Ⅳ期垂体腺瘤患者(0.473±0.060)垂体腺瘤组织的HCG4水平均低于正常组织(P<0.05)。HCG4组的β-catenin蛋白相对表达量低于NC组(P<0.05)。HCG4组HP75细胞的增殖、迁移和侵袭能力均弱于NC组(P<0.05);HCG4+Wnt激动剂组HP75细胞的增殖、迁移和侵袭能力均强于HCG4组(P<0.05)。另外,HCG4组β-catenin和MMP-9水平低于NC组(P<0.05);HCG4+Wnt激动剂组的β-catenin和MMP-9水平高于HCG4组(P<0.05)。结论HCG4通过抑制Wnt/β-catenin信号通路活性参与垂体腺瘤细胞增殖、迁移和侵袭过程来抑制垂体腺瘤恶性发展,可能为垂体瘤治疗提供潜在新靶点。

Objective To investigate the effects of long non-coding RNA HLA complex group 4(HCG4)on proliferation,migration and invasion process of pituitary adenoma cell through Wnt/β-catenin signaling pathway.Methods Real time-quantitative polymerase chain reaction(qPCR)was employed to access the levels of HCG4 in 35 paired normal pituitary glands and pituitary adenoma tissues.Pituitary adenoma HP75 cells were divided into Control group(untreated cells),NC group(cells transfected with empty plasmid pcDNA3.1),HCG4 group(cells transfected with overexpression plasmid pcDNA3.1-HCG4)and HCG4+Wnt activator group(cells transfected with pcDNA3.1-HCG4 and challenged with Wnt signal activator SKL2001).The CCK-8,scratch and Transwell chamber assays were applied to detect the proliferation,migration and invasion abilities of HP75 cell.QPCR and Western blotting were applied to determine the expressions ofβ-catenin and matrix metalloproteinase(MMP)-9.Results The levels of HCG4 in pituitary adenoma tissues were lower than those of normal pituitary glands(0.616±0.044 vs.1.000±0.071,P<0.05).HCG4 levels were lower in 21 patients with stageⅠ–Ⅱ(0.712±0.053,n=21)and stageⅢ–Ⅳ(0.473±0.060,n=14)than those of normal pituitary glands(P<0.05).Levels ofβ-catenin in HCG4 group were declined as compared to NC group(P<0.05).The abilities of proliferation,migration and invasion of HP75 cells were weakened in HCG4 group as compared to NC group(P<0.05).The abilities of proliferation,migration and invasion of HP75 cells were enhanced in HCG4+Wnt activator group as compared to HCG4 group(P<0.05).Meanwhile,the levels ofβ-catenin and MMP-9 in HCG4 group were lower than NC group(P<0.05).The levels ofβ-catenin and MMP-9 in HCG4+Wnt activator group were higher than those in HCG4 group(P<0.05).Conclusion HCG4 inhibited pituitary adenoma development by participating in cell proliferation,migration and invasion process through suppressing Wnt/β-catenin signaling pathway,which may present a novel target for pituitary tumor treatment.